Background/Purpose:

Angiogenesis is regarded as a switch from acute to chronic inflammation and thus plays a crucial role in rheumatoid arthritis disease progression. This process is highly complex, involving interactions between several cell types in the inflamed synovium. Many of the current in vitro models of angiogenesis focus solely on endothelial cells (EC) and do not include interactions with other cell types. RA synovial fibroblasts (RASF) are important contributors to angiogenesis in synovial inflammation, and therefore a model including both RASF and EC would more accurately represent the pathophysiology of RA angiogenesis. We previously demonstrated that NF-κB inducing kinase (NIK) dependent non-canonical NF-κB signaling can induce angiogenesis in EC. This has not yet been studied in a co-culture model of angiogenesis.

Objective:

To develop a 3D in vitro model to study the interaction between RASF and EC and to further delineate the role of the non-canonical NF-κB pathway in pathological angiogenesis. 

Methods:

We developed a novel 3 dimensional (3D) model in which human umbilical cord vein EC and RASF were labeled with green or orange cell tracker dye, respectively, and incubated overnight to form spheroids. Subsequently, the spheroids were harvested and plated in a collagen solution, and medium with or without lymphotoxin α1β2 (LT) or LIGHT (both stimuli that induce non-canonical NF-κB signaling via the lymphotoxin beta receptor (LTβR)) or pro-angiogenic growth factors (bFGF/VEGF) was added. After 48 hours, spheroids were fixed and imaged through confocal microscopy. Cumulative EC sprout length and the number of sprouts was quantified using Leica QWin Plus software. To demonstrate NIK dependency of this process, EC were transfected with non-targeting or NIK targeting siRNA before addition into the model and subsequent sprout formation was quantified.

Results:

Confocal analysis of the 3D model showed spheroids containing HUVEC and RASF formed sprouts under all conditions. Both  LT and LIGHT caused significant increases in cumulative sprout length (p<0.05). Interestingly, the total number of sprouts formed by each spheroid also increased significantly. LT and LIGHT induced sprout formation was significantly decreased by siRNA-mediated knockdown of NIK in EC as compared to the non-targeting siRNA controls. 

Conclusion:

Our novel 3D model demonstrates that activation of the non-canonical pathway induces angiogenesis in spheroids of EC and RASF and that this process is strictly NIK dependent.  This suggests that NIK targeting therapeutics may be able to reduce pathological angiogenesis in synovial inflammation and possibly halt disease progression. Further studies to test this, including the use of small molecule pharmacological NIK inhibitors, are currently underway. Of interest, the current 3D model is also optimized to include different subsets of immune cells in order to study their contributions to inflammation-induced angiogenesis, which makes this model a valuable tool for future studies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/non-canonical-nf-kappab-signaling-promotes-angiogenesis-in-a-novel-3d-spheroid-model-of-rheumatoid-arthritis-synovial-inflammation/